Protein In Xenopus Oocytes Research Articles

Species variants of the I sK protein: differences in kinetics, voltage dependence, and La3+ block of the currents expressed in Xenopus oocytes

We have compared the slowly activating K+ currents (IsK) resulting from the expression of the human, mouse, or rat IsK proteins in Xenopus oocytes, utilizing natural, species-dependent sequence variations to initiate structure-function studies of this channel. Differences were found between the human and rodent currents in their voltage dependence, kinetics, and sensitivity to external La3+. The current/voltage relationships of the human and rat IsK currents differed significantly, with greater depolarizations required for activation of the human channel. The first 30 s of activation during depolarizations to potentials between -10 and +40 mV was best described by a triexponential function for each of the three species variants. The activation rates were, however, significantly faster for the human current than for either of the rodent forms. Similarly, deactivation kinetics were best described as a biexponential decay for each of the species variants but the human currents deactivated more rapidly than the rodent currents. The human and the rodent forms of IsK were also differentially affected by external La3+. Low concentrations (10, 50 microM) rapidly and reversibly reduced the magnitude of the mouse and rat currents during a test depolarization and increased the deactivation rates of the tail currents. In contrast, the magnitude and deactivation rates of the human IsK currents were unaffected by 50 microM La3+.

Endogenous amino acid transport systems and expression of mammalian amino acid transport proteins in Xenopus oocytes

Oocyte amino acid transport has physiological significance to oocytes and practical importance to molecular biologists and transport physiologists. Expression of heterologous mRNA in Xenopus oocytes is currently being used to help clone cDNAs for amino acid transporters and their effectors. A major question to be resolved in many of these studies is whether the injected mRNA codes for a transporter or an activator of an endogenous system. Nevertheless, the cDNAs of several families of amino acid transporters or their activators appear already to have been cloned. One such transporter is the anion exchanger, band 3, which may also transport glycine and taurine under some important physiological conditions such as hypoosmotic stress. Site-directed mutagenesis of band 3 has already shown that an amino acid residue believed to be at or near the active site nevertheless does not appear to influence Cl- transport in Xenopus oocytes expressing the modified band 3 protein. Continuation of such studies along with examination of transport of all possible substrates of band 3 should yield insight into the relationship between the structure and function of this transporter. Each of three other families not only contains amino acid transporters, but also appears to contain members that serve as transporters of neurotransmitters or their metabolites. Because of the distinct structural differences in the preferred substrates of different transporters within some of these families, elucidation of the tertiary and possibly quaternary structural relationships among the members of such families may reveal transport mechanisms. In addition, the grouping of neurotransmitters or their metabolites according to the family to which their transport systems and transporters belong could yield insight into mechanisms of brain development, function and evolution. Another family of transporters for cationic amino acids also serves, at least in one case, as a viral receptor. Hence, these or other transporters also could conceivably function in eggs as receptors for sperm and, more broadly, in cell-cell interactions as well as in amino acid transport. Moreover, a family of apparent amino acid transport activators are homologous to a family of glycosidases, so these activators could also serve to recognize carbohydrate structures on other cells or the extracellular matrix. Some of these activators appear to increase more than one amino acid transport activity in Xenopus oocytes. In other studies, expression of heterologous mRNA in oocytes has led apparently to detection of inhibitors as well as activators of amino acid transport. Some amino acid transport systems also could conceivably contain nucleic acid as well as glycoprotein components.(ABSTRACT TRUNCATED AT 400 WORDS)

N-linked glycosylation of the receptor for murine ecotropic retroviruses is altered in virus-infected cells

Pulse-chase labeling identified incomplete maturation of the cationic amino acid transporter that serves as the receptor for ecotropic retroviruses in infected NIH 3T3 fibroblasts. The molecular basis and functional consequences of this change were studied by expressing viral and receptor proteins in Xenopus oocytes. Expression of the ecotropic, but not polytropic, envelope surface protein, gp70, interfered with N-linked glycosylation of the permissive mouse, but not the nonpermissive, human transporter. Incomplete glycosylation may be a direct consequence of gp70 binding since the glycosylated residues, Asn223 and Asn229, are present in the envelope binding domain. Receptors bearing amino acid substitutions in Asn223 and/or Asn229 that prevent glycosylation function normally in virus infection and amino acid transport. However, gp70 expression sufficient to block binding of 125I-gp70 to the receptor in the plasma membrane decreased receptor-mediated arginine uptake by 50%. Taken together, these findings suggest that newly synthesized gp70 may bind to the receptor in the endoplasmic reticulum and prevent normal glycosylation during their transit to the plasma membrane.

Two upstream cysteines and the CAAX motif but not the polybasic domain are required for membrane association of Xlcaax in Xenopus oocytes.

We have analyzed the role of several protein motifs in controlling the membrane association of the xlcaax-1 protein in Xenopus oocytes. Xlcaax-1 is a maternally expressed protein that during development is associated with the basal lateral membrane of polarized epithelial cells. It is enriched in the tubule cells of the adult kidney and several other organs that are involved in osmoregulation. Xlcaax-1 has a C-terminal CAAX sequence (CVVM) identical to that of N-ras, followed by two cysteines that are potential palmitoylation sites and a polybasic domain. Mutants were constructed that either deleted specific domains or changed specific amino acids of the consensus sequences in or near the CAAX motif. Synthetic mRNAs were injected into Xenopus oocytes and their protein products analyzed for their ability to associate with the oocyte plasma membrane. A mutation changing cysteine-588 of the CAAX box to serine or the inhibition of prenylation by lovastatin eliminated the membrane association of the protein. Mutation of either of the upstream cysteines (either 585 or 587) also inhibited the association of xlcaax-1 with the membrane. Unlike Ras, however, deletion of the polybasic domain had no effect on membrane binding. In addition, we show that xlcaax-1 binds ATP but not GTP.

The vacuolar membrane protein gamma-TIP creates water specific channels in Xenopus oocytes.

The vacuolar membrane (tonoplast) of higher plant cells contains an abundant 27 kDa protein called TIP (tonoplast intrinsic protein) that occurs in different isoforms and belongs to a large family of homologous channel-like proteins found in bacteria, plants and animals. In the present study, we identified and characterized the function of gamma-TIP from Arabidopsis thaliana by expression of the protein in Xenopus oocytes. gamma-TIP increased the osmotic water permeability of oocytes 6- to 8-fold, to values in the range 1-1.5 x 10(-2) cm/s. Similar results were obtained with the homologous human erythrocyte protein CHIP28, recently identified as the erythrocyte water channel. The bacterial homolog GlpF did not affect the osmotic water permeability of oocytes, but facilitated glycerol uptake, in accordance with its known function. By contrast, gamma-TIP did not promote glycerol permeability. Voltage clamp experiments provided evidence showing that gamma-TIP induced no electrogenic ion transport in oocytes, especially during osmotic challenge that resulted in massive transport of water. These results allow us to conclude that the various protein members of the MIP family have unique and specific transport functions and that the plant protein gamma-TIP likely functions as a water specific channel in the vacuolar membrane.

The role of nucleoplasmin in chromatin assembly and disassembly

Nucleoplasmin is the most abundant nuclear protein in Xenopus oocytes and eggs. The term ‘molecular chaperone’ was coined to describe its role in the assembly of the nucleosome subunits of chromatin. Although histones and DNA can self-assemble into nucleosomes, nucleoplasmin can facilitate this process in vitro by competing against non-specific charge interactions. In vivo nucleoplasmin binds histones H2A and H2B and transfers them to DNA. Another acidic nuclear protein, N l, binds and transfers histones H3 and H4. Nucleoplasmin has at least one other role in modulating chromatin structure in Xenopus eggs. It is required for the first stage of sperm chromatin decondensation. It binds and removes sperm basic proteins and replaces them by histones H2A and H2B, again forming nucleosomes, and resulting in decondensation of the compacted sperm chromatin. In addition we propose that the properties of the nuclear localization signal of nucleoplasmin can be explained by a model in which heat shock cognate protein hsc70 has a chaperone role in signal presentation during nuclear transport.

A Possible Role for a Mammalian Facilitative Hexose Transporter in the Development of Resistance to Drugs

We show that D- but not L-hexoses modulate the accumulation of radioactive vinblastine in injected Xenopus laevis oocytes expressing the murine Mdr1b P-glycoprotein. We also show that X. laevis oocytes injected with RNA encoding the rat erythroid/brain glucose transport protein (GLUT1) and expressing the corresponding functional transporter exhibit a lower accumulation of [3H]vinblastine and show a greater capacity to extrude the drug than do control oocytes not expressing the rat GLUT1 protein. Cytochalasin B and phloretin, two inhibitors of the mammalian facilitative glucose transporters, can overcome the reduced drug accumulation conferred by expression of the rat GLUT1 protein in Xenopus oocytes but have no significant effect on the accumulation of drug by Xenopus oocytes expressing the mouse Mdr1b P-glycoprotein. These drugs also increase the accumulation of [3H]vinblastine in multidrug-resistant Chinese hamster ovary cells. Cytochalasin E, an analog of cytochalasin B that does not affect the activity of the facilitative glucose transporter, has no effect on the accumulation of vinblastine by multidrug-resistant Chinese hamster cells or by oocytes expressing either the mouse Mdr1b P-glycoprotein or the GLUT1 protein. In all three cases, the drug verapamil produces a profound effect on the cellular accumulation of vinblastine. Interestingly, although immunological analysis indicated the presence of massive amounts of P-glycoprotein in the multidrug-resistant cells, immunological and functional studies revealed only a minor increase in the expression of a hexose transporter-like protein in resistant versus drug-sensitive cells. Taken together, these results suggest the participation of the mammalian facilitative glucose transporter in the development of drug resistance.

A possible role for a mammalian facilitative hexose transporter in the development of resistance to drugs.

We show that D- but not L-hexoses modulate the accumulation of radioactive vinblastine in injected Xenopus laevis oocytes expressing the murine Mdr1b P-glycoprotein. We also show that X. laevis oocytes injected with RNA encoding the rat erythroid/brain glucose transport protein (GLUT1) and expressing the corresponding functional transporter exhibit a lower accumulation of [3H]vinblastine and show a greater capacity to extrude the drug than do control oocytes not expressing the rat GLUT1 protein. Cytochalasin B and phloretin, two inhibitors of the mammalian facilitative glucose transporters, can overcome the reduced drug accumulation conferred by expression of the rat GLUT1 protein in Xenopus oocytes but have no significant effect on the accumulation of drug by Xenopus oocytes expressing the mouse Mdr1b P-glycoprotein. These drugs also increase the accumulation of [3H]vinblastine in multidrug-resistant Chinese hamster ovary cells. Cytochalasin E, an analog of cytochalasin B that does not affect the activity of the facilitative glucose transporter, has no effect on the accumulation of vinblastine by multidrug-resistant Chinese hamster cells or by oocytes expressing either the mouse Mdr1b P-glycoprotein or the GLUT1 protein. In all three cases, the drug verapamil produces a profound effect on the cellular accumulation of vinblastine. Interestingly, although immunological analysis indicated the presence of massive amounts of P-glycoprotein in the multidrug-resistant cells, immunological and functional studies revealed only a minor increase in the expression of a hexose transporter-like protein in resistant versus drug-sensitive cells. Taken together, these results suggest the participation of the mammalian facilitative glucose transporter in the development of drug resistance.

Tyrosine Phosphorylation and Activation of Homologous Protein Kinases during Oocyte Maturation and Mitogenic Activation of Fibroblasts

Meiotic maturation of Xenopus and sea star oocytes involves the activation of a number of protein-serine/threonine kinase activities, including a myelin basic protein (MBP) kinase. A 44-kDa MBP kinase (p44mpk) purified from mature sea star oocytes is shown here to be phosphorylated at tyrosine. Antiserum to purified sea star p44mpk was used to identify antigenically related proteins in Xenopus oocytes. Two tyrosine-phosphorylated 42-kDa proteins (p42) were detected with this antiserum in Xenopus eggs. Xenopus p42 chromatographs with MBP kinase activity on a Mono Q ion-exchange column. Tyrosine phosphorylation of Xenopus p42 approximately parallels MBP kinase activity during meiotic maturation. These results suggest that related MBP kinases are activated during meiotic maturation of Xenopus and sea star oocytes. Previous studies have suggested that Xenopus p42 is related to the mitogen-activated protein (MAP) kinases of culture mammalian cells. We have cloned a MAP kinase relative from a Xenopus ovary cDNA library and demonstrate that this clone encodes the Xenopus p42 that is tyrosine phosphorylated during oocyte maturation. Comparison of the sequences of Xenopus p42 and a rat MAP kinase (ERK1) and peptide sequences from sea star p44mpk indicates that these proteins are close relatives. The family members appear to be tyrosine phosphorylated, and activated, in different contexts, with the murine MAP kinase active during the transition from quiescence to the G1 stage of the mitotic cell cycle and the sea star and Xenopus kinases being active during M phase of the meiotic cell cycle.

Tyrosine phosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts.

Meiotic maturation of Xenopus and sea star oocytes involves the activation of a number of protein-serine/threonine kinase activities, including a myelin basic protein (MBP) kinase. A 44-kDa MBP kinase (p44mpk) purified from mature sea star oocytes is shown here to be phosphorylated at tyrosine. Antiserum to purified sea star p44mpk was used to identify antigenically related proteins in Xenopus oocytes. Two tyrosine-phosphorylated 42-kDa proteins (p42) were detected with this antiserum in Xenopus eggs. Xenopus p42 chromatographs with MBP kinase activity on a Mono Q ion-exchange column. Tyrosine phosphorylation of Xenopus p42 approximately parallels MBP kinase activity during meiotic maturation. These results suggest that related MBP kinases are activated during meiotic maturation of Xenopus and sea star oocytes. Previous studies have suggested that Xenopus p42 is related to the mitogen-activated protein (MAP) kinases of culture mammalian cells. We have cloned a MAP kinase relative from a Xenopus ovary cDNA library and demonstrate that this clone encodes the Xenopus p42 that is tyrosine phosphorylated during oocyte maturation. Comparison of the sequences of Xenopus p42 and a rat MAP kinase (ERK1) and peptide sequences from sea star p44mpk indicates that these proteins are close relatives. The family members appear to be tyrosine phosphorylated, and activated, in different contexts, with the murine MAP kinase active during the transition from quiescence to the G1 stage of the mitotic cell cycle and the sea star and Xenopus kinases being active during M phase of the meiotic cell cycle.

Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes

A complementary DNA encoding a voltage-gated chloride channel from Torpedo marmorata electric organ was cloned by expressing hybrid-depleted messenger RNA in Xenopus oocytes. The predicted protein has a sequence of 805 amino acids containing several putative membrane-spanning domains. Expression of the protein in Xenopus oocytes shows that it is sufficient for channel function.

Activation of chromosomal vitellogenin genes in Xenopus oocytes by pure estrogen receptor and independent activation of albumin genes.

We generate pure estrogen receptor protein in Xenopus oocytes by injecting them with estrogen receptor mRNA synthesized in vitro. A chromosomal vitellogenin gene, which normally responds to estrogen only in liver cells, is activated. Primer extension shows that initiation is accurate, and ribonuclease mapping shows that the first exon is correctly spliced out of the initial transcript. Long transcripts are produced, one being equal in length to poly(A)- vitellogenin mRNA. Immunochemical estimates of receptor levels in the oocyte nuclei suggest that pure receptor, acting alone, cannot activate oocyte vitellogenin genes unless unusually large amounts are present. However, when a receptor-free extract from liver cells is also injected, the amount of receptor required is reduced. Such an extract, but not pure receptor, can also activate albumin genes in oocytes.

Activation of Chromosomal Vitellogenin Genes in Xenopus Oocytes by Pure Estrogen Receptor and Independent Activation of Albumin Genes

We generate pure estrogen receptor protein in Xenopus oocytes by injecting them with estrogen receptor mRNA synthesized in vitro. A chromosomal vitellogenin gene, which normally responds to estrogen only in liver cells, is activated. Primer extension shows that initiation is accurate, and ribonuclease mapping shows that the first exon is correctly spliced out of the initial transcript. Long transcripts are produced, one being equal in length to poly(A)- vitellogenin mRNA. Immunochemical estimates of receptor levels in the oocyte nuclei suggest that pure receptor, acting alone, cannot activate oocyte vitellogenin genes unless unusually large amounts are present. However, when a receptor-free extract from liver cells is also injected, the amount of receptor required is reduced. Such an extract, but not pure receptor, can also activate albumin genes in oocytes.

Prenylation of mammalian Ras protein in Xenopus oocytes.

Ras protein requires an intermediate of the cholesterol biosynthetic pathway for posttranslational modification and membrane anchorage. This step is necessary for biological activity. Maturation of Xenopus laevis oocytes induced by an oncogenic human Ras protein can be inhibited by lovastatin or compactin, inhibitors of the synthesis of mevalonate, an intermediate of cholesterol biosynthesis. This inhibition can be overcome by mevalonic acid or farnesyl diphosphate, a cholesterol biosynthetic intermediate downstream of mevalonate, but not by squalene, an intermediate after farnesyl pyrophosphate in the pathway. This study supports the idea that in Xenopus oocytes, the Ras protein is modified by a farnesyl moiety or its derivative. Furthermore, an octapeptide with the sequence similar to the C-terminus of the c-H-ras protein inhibits the biological activity of Ras proteins in vivo, suggesting that it competes for the enzyme or enzymes responsible for transferring the isoprenoid moiety (prenylation) in the oocytes. This inhibition of Ras prenylation by the peptide was also observed in vitro, using both Saccharomyces cerevisiae and Xenopus oocyte extracts. These observations show that Xenopus oocytes provide a convenient in vivo system for studies of inhibitors of the posttranslational modification of the Ras protein, especially for inhibitors such as peptides that do not penetrate cell membranes.

Expression of the brown fat mitochondria uncoupling protein in Xenopus oocytes and import into mitochondrial membrane

Non shivering thermogenesis of brown adipose tissue is due to the uncoupling protein (UCP), located in the inner mitochondrial membrane, which functions as a proton translocator and can thus uncouple mitochondrial respiration. We describe here the expression of UCP in Xenopus laevis oocytes after injection of UCP mRNA, which was transcribed in vitro. UCP seems to be correctly transported into mitochondria and integrated into the membrane, but we were not able to establish definitely the functionality of this UCP. We conclude that this expression system could be suitable for the study of the mitochondrial import mechanism but not for the examination of physiological properties of UCP.

Photocrosslinking of proteins to maternal mRNA in Xenopus oocytes

Ultraviolet irradiation was used to covalently crosslink poly(A) RNA and associated proteins in Xenopus oocytes and reticulocytes. Each cell type contained similar as well as unique crosslinked proteins. The somatic cells contained a single 78-kDa 3′ poly(A) tract binding protein while oocyte poly(A), however, was bound by this protein and at least three additional proteins. Based on the mass of poly(A) RNA, oocytes in their earliest stages of growth contained crosslinked proteins that were generally more prevalent than in fully grown oocytes. An investigation of possible messenger RNA-specific proteins was undertaken by a series of RNA injection experiments. Two radiolabeled SP6-derived mRNAs were injected into oocytes; the first, globin mRNA, assembled into polysomes, while the second, a maternal mRNA termed G10, entered a nontranslating ribonucleoprotein compartment. Following the induction of oocyte maturation, additional globin mRNA was recruited onto polysomes while G10 mRNA remained a nontranslating mRNP. The proteins that can be crosslinked to these injected mRNAs were detected by 32P nucleotide transfer. Each mRNA associated with shared as well as unique proteins, some of which were detected only in mature oocytes. The possible function of these proteins is discussed.

Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP78 in Xenopus oocytes.

We have studied the compartmentation and movement of the rat 78-kd glucose-regulated protein (GRP78) and other secretory and membrane proteins in Xenopus oocytes. Full length GRP78, normally found in the lumen of rat endoplasmic reticulum (ER), is localized to a membraneous compartment in oocytes and is not secreted. A truncated GRP78 lacking the C-terminal (KDEL) ER retention signal is secreted, although at a slow rate. When the synthesis of radioactive GRP78 is confined to a polar (animal or vegetal) region of the oocyte and the subsequent movement across the oocyte monitored, we find that both full-length and truncated GRP78 move at similar rates and only slightly slower than a secretory protein, chick ovalbumin. In contrast, a plasma membrane protein (influenza haemagglutinin) and two ER membrane proteins (rotavirus VP10 and a mutant haemagglutinin) remained confined to their site of synthesis. We conclude that the retention of GRP78 in the ER is not due to its tight binding to a membrane-bound receptor.

Molecular cloning, full-length sequence and preliminary characterization of a 56-kDa protein induced by human interferons.

Among the various proteins which are induced when human cells are treated with interferon, a predominant protein of unknown function, with molecular mass 56 kDa, has been observed. With the aim of exploring the molecular basis of the regulation of this protein and of its mRNA, in order to understand its biological function and its possible contribution to the various antiviral and non-antiviral actions exerted by interferons, we cloned a full-length cDNA copy of the 1.8-1.9 X 10(3)-base 56-kDa-protein mRNA and determined its sequence. The cDNA contains 1662 nucleotides derived from the 56-kDa-protein mRNA, including a poly(A) tail of 20 residues. Primer extension experiments indicate that the 5' end of this cDNA clone is probably located only 13 nucleotides downstream of the actual cap site of the 56-kDa-protein mRNA. It consists of a 64-nucleotide-long 5'-non-coding segment, a coding segment of 1434 nucleotides terminated by a TAG triplet and a 141-nucleotide 3'-non-coding segment. The encoded protein of 478 amino acid residues has a molecular mass of 55335 Da and a single potential site for N-glycosylation. The protein contains an excess of basic amino acids and most of them are localized in the carboxy-terminal half of the molecule. A single [35S]methionine-labeled 56-kDa protein was obtained using an SP64 construction to allow the cell-free transcription and translation of the cloned cDNA. Microinjection of this labeled protein in Xenopus oocytes indicates that the 56-kDa protein is cytoplasmic.

Stability and movement of mRNAs and their encoded proteins in Xenopus oocytes.

The stability and movement of several polyadenylated (poly A+) and nonpolyadenylated (poly A-) mRNAs in Xenopus oocytes have been examined. At least 50% of the poly A+ mRNA molecules (9S rabbit globin mRNA, chicken ovalbumin, and lysozyme) were stable in oocytes over a 48-h period, irrespective of the amount injected. About 50% of injected poly A- reovirus mRNAs was degraded within the first 24 h of injection, irrespective of the amount injected, although no further degradation was observed over an additional 24 h. The movement of all poly A+ mRNAs injected at either the animal or vegetal pole of the oocyte was very slow. Little movement of RNA from the animal half to the vegetal half was observed even 48 h after injection. In contrast, similar amounts of mRNA were present in both halves 48 h after vegetal pole injection. Similar results were obtained after injection of poly A- reovirus mRNAs. The movement of the proteins encoded by the poly A+ mRNAs was studied in the 6-h period after injection when little mRNA movement had occurred. 85% of the globin synthesized accumulated in the animal half irrespective of injection site. The movement of the sequestered secretory proteins ovalbumin and lysozyme in the same oocytes as globin was much slower; very little lysozyme appeared in the half of the oocyte opposite the site of injection.

Segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in Xenopus oocytes : Identification of an ovalbumin signal sequence

The intramolecular signals for chicken ovalbumin secretion were examined by producing mutant proteins in Xenopus oocytes. An ovalbumin complementary DNA clone was manipulated in vitro, and constructs containing altered protein-coding sequences and either the simian virus 40 (SV40) early promoter or Herpes simplex thymidine kinase promoter, were microinjected into Xenopus laevis oocytes. The removal of the eight extreme N-terminal amino acids of ovalbumin had no effect on the segregation of ovalbumin with oocyte membranes nor on its secretion. A protein lacking amino acids 2 to 21 was sequestered in the endoplasmic reticulum but remained strongly associated with the oocyte membranes rather than being secreted. Removal of amino acids 231 to 279, a region previously reported to have membrane-insertion function, resulted in a protein that also entered the endoplasmic reticulum but was not secreted. Hybrid proteins containing at their N terminus amino acids 9 to 41 or 22 to 41 of ovalbumin fused to the complete chimpanzee α-globin polypeptide were also sequestered by oocyte membranes. We conclude that the ovalbumin “signal” sequeǹce is internally located within amino acids 22 to 41, and we speculate that amino acids 9 to 21 could be important for the completion of ovalbumin translocation through membranes.